Integrative framework for identification of key cell identity genes uncovers determinants of ES cell identity and homeostasis.

Identification of genes associated with specific biological phenotypes is a fundamental step toward understanding the molecular basis underlying development and pathogenesis. Although RNAi-based high-throughput screens are routinely used for this task, false discovery and sensitivity remain a challenge. Here we describe a computational framework for systematic integration of published gene expression data to identify genes defining a phenotype of interest. We applied our approach to rank-order all genes based on their likelihood of determining ES cell (ESC) identity. RNAi-mediated loss-of-function experiments on top-ranked genes unearthed many novel determinants of ESC identity, thus validating the derived gene ranks to serve as a rich and valuable resource for those working to uncover novel ESC regulators. Underscoring the value of our gene ranks, functional studies of our top-hit Nucleolin (Ncl), abundant in stem and cancer cells, revealed Ncl's essential role in the maintenance of ESC homeostasis by shielding against differentiation-inducing redox imbalance-induced oxidative stress. Notably, we report a conceptually novel mechanism involving a Nucleolin-dependent Nanog-p53 bistable switch regulating the homeostatic balance between self-renewal and differentiation in ESCs. Our findings connect the dots on a previously unknown regulatory circuitry involving genes associated with traits in both ESCs and cancer and might have profound implications for understanding cell fate decisions in cancer stem cells. The proposed computational framework, by helping to prioritize and preselect candidate genes for tests using complex and expensive genetic screens, provides a powerful yet inexpensive means for identification of key cell identity genes.

Acute depletion of Tet1-dependent 5-hydroxymethylcytosine levels impairs LIF/Stat3 signaling and results in loss of embryonic stem cell identity.

The TET family of FE(II) and 2-oxoglutarate-dependent enzymes (Tet1/2/3) promote DNA demethylation by converting 5-methylcytosine to 5-hydroxymethylcytosine (5hmC), which they further oxidize into 5-formylcytosine and 5-carboxylcytosine. Tet1 is robustly expressed in mouse embryonic stem cells (mESCs) and has been implicated in mESC maintenance. Here we demonstrate that, unlike genetic deletion, RNAi-mediated depletion of Tet1 in mESCs led to a significant reduction in 5hmC and loss of mESC identity. The differentiation phenotype due to Tet1 depletion positively correlated with the extent of 5hmC loss. Meta-analyses of genomic data sets suggested interaction between Tet1 and leukemia inhibitory factor (LIF) signaling. LIF signaling is known to promote self-renewal and pluripotency in mESCs partly by opposing MAPK/ERK-mediated differentiation. Withdrawal of LIF leads to differentiation of mESCs. We discovered that Tet1 depletion impaired LIF-dependent Stat3-mediated gene activation by affecting Stat3's ability to bind to its target sites on chromatin. Nanog overexpression or inhibition of MAPK/ERK signaling, both known to maintain mESCs in the absence of LIF, rescued Tet1 depletion, further supporting the dependence of LIF/Stat3 signaling on Tet1. These data support the conclusion that analysis of mESCs in the hours/days immediately following efficient Tet1 depletion reveals Tet1's normal physiological role in maintaining the pluripotent state that may be subject to homeostatic compensation in genetic models.

Cnot1, Cnot2, and Cnot3 maintain mouse and human ESC identity and inhibit extraembryonic differentiation.

Embryonic stem cell (ESC) identity and self-renewal is maintained by extrinsic signaling pathways and intrinsic gene regulatory networks. Here, we show that three members of the Ccr4-Not complex, Cnot1, Cnot2, and Cnot3, play critical roles in maintaining mouse and human ESC identity as a protein complex and inhibit differentiation into the extraembryonic lineages. Enriched in the inner cell mass of blastocysts, these Cnot genes are highly expressed in ESC and downregulated during differentiation. In mouse ESCs, Cnot1, Cnot2, and Cnot3 are important for maintenance in both normal conditions and the 2i/LIF medium that supports the ground state pluripotency. Genetic analysis indicated that they do not act through known self-renewal pathways or core transcription factors. Instead, they repress the expression of early trophectoderm (TE) transcription factors such as Cdx2. Importantly, these Cnot genes are also necessary for the maintenance of human ESCs, and silencing them mainly lead to TE and primitive endoderm differentiation. Together, our results indicate that Cnot1, Cnot2, and Cnot3 represent a novel component of the core self-renewal and pluripotency circuitry conserved in mouse and human ESCs.

Cnot3 is required for male germ cell development and spermatogonial stem cell maintenance.

The foundation of spermatogenesis and lifelong fertility is provided by spermatogonial stem cells (SSCs). SSCs divide asymmetrically to either replenish their numbers (self-renewal) or produce undifferentiated progenitors that proliferate before committing to differentiation. However, regulatory mechanisms governing SSC maintenance are poorly understood. Here, we show that the CCR4-NOT mRNA deadenylase complex subunit CNOT3 plays a critical role in maintaining spermatogonial populations in mice. Cnot3 is highly expressed in undifferentiated spermatogonia, and its deletion in spermatogonia resulted in germ cell loss and infertility. Single cell analyses revealed that Cnot3 deletion led to the de-repression of transcripts encoding factors involved in spermatogonial differentiation, including those in the glutathione redox pathway that are critical for SSC maintenance. Together, our study reveals that CNOT3 - likely via the CCR4-NOT complex - actively degrades transcripts encoding differentiation factors to sustain the spermatogonial pool and ensure the progression of spermatogenesis, highlighting the importance of CCR4-NOT-mediated post-transcriptional gene regulation during male germ cell development.